This revised renewal application is directed toward understanding the multifaceted role of the c-myc oncogene in human breast cancer. Our proposal focuses on elucidation of the mechanisms whereby c-Myc protein causes malignant behavior of human mammary epithelial cells. In a coordinated, translational research proposal, experiments will be conducted with primary and immortalized human mammary epithelial cell (HMEC) cultures in vitro and with archival human breast tumor specimens. In Aim 1, we will determine how c-Myc-cell adhesion interactions regulate cell transformation (anchorage independent proliferation) in human mammary epithelial cells. We have shown that in immortalized HMECs, deregulated expression of c- Myc allows anchorage independent, G1/S cell cycle progression, closely associated with proteosome-mediated degradation of p27. We now use a newly established, regulatable MCF-10A-c-MycER system and non-immortalized HMEC to elucidate the mechanism and relevance for cell transformation of this effect. Experiments will determine the roles of SCFSKP2 ubiquitin ligase components, MAP kinases, and Jab1-dependent cytoplasmic localization of p27, in c-Myc triggered destruction of p27. In addition, experiments will establish downstream consequences of p27 destabilization for activation of CDK2 and inactivation of pRb for induction of anchorage independent cell proliferation. In Aim 2, we will seek to confirm that the c-Myc-dependent mechanisms, observed in Aim 1 are relevant to human breast cancer. This second Aim is based on our demonstration that c-myc gene amplification correlates with its expression, commonly occurring in human breast cancer. In the previous grant period we reported a meta-analysis of the literature that strongly suggested that c-myc gene amplification is significantly associated with high grade, progesterone receptor negative disease (irrespective of estrogen receptor status). Importantly, c-myc amplification was significantly correlated with lymph node metastasis and poor disease free and overall survival. In Aim 2, we will first determine whether deregulated expression of c-Myc preceeds its gene amplification, in the natural history of the disease. Second, we will determine whether tumor proliferation is correlated positively with c-Myc protein expression, negatively with p27, and positively with expression of mediators of p27 degradation: SCF SKP2 ubiquitin ligase and Jab1. Third, we will determine whether tumor apoptosis is correlated with proliferation, c-Myc and wild type p53. Finally, we will determine whether c-Myc amplification or expression is more closely correlated with poor clinical outcome. Thus, our new proposal builds on our prior funding period to further examine mechanisms of c-Myc-promoted malignancy in the human mammary epithelial cell in vitro and in human breast cancer in women.